The present invention relates to immunomodulatory compositions and methods.
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Cytokines and related immunomodulatory compounds play an important role in the regulation and function of the immune system, making them suitable targets for therapeutic intervention in diseases involving immune system dysfunction. It would therefore be desirable to identify heretofore undiscovered genes encoding cytokines and other immunomodulatory compounds, which may be useful as a basis for treatment of diseases affecting or influenced by the immune system. Present methods for the identification of such genes have met with limited success. These methods include (i) screening for DNAse I hypersensitive sites and HTF islands as potential markers for transcription units, (ii) cross-species hybridization analysis of genomic sequences, (iii) hybridization of radiolabelled cDNAs to arrayed genomic clones, (iv) screening of cDNA libraries with complex genomic probes, (v) exon trapping, (vi) random sequencing and assignment of tissue-specific cDNAs, (vii) xe2x80x9csoftware trappingxe2x80x9d of the genes in extensive genomic sequencing projects, and (viii) cDNA normalization, subtraction or/and hybridization selection using extensive genomic fragments.
Most of the above approaches have proven either unreliable, or have required a substantial effort to find the genes of interest. For instance, a conventional xe2x80x9cfunctionalxe2x80x9d gene cloning route includes purifying the protein factor with a particular biological activity, microsequencing the protein to design a redundant oligoprobe, raising antibodies to the protein, expression cloning of the candidate gene or conventional screening of cDNA libraries with the redundant probe.
En masse cDNA sequencing efforts have contributed substantially to novel gene discovery by identifying a large number of novel sequences and tissue expression xe2x80x9cprofilesxe2x80x9d. However, because these efforts typically had no defined targets and depended on screening conventional cDNA libraries, they resulted in the preferential identification of common, abundant cDNAs, and were thus biased against the identification of novel cytokine genes, which tend to be selectively expressed at relatively low levels.
Exon trapping can be efficiently used to screen complex genomic DNA. This method is widely-used due to its independence of the gene expression in any particular cell line or tissue, but it requires substantial further efforts for isolation and identification of the genes in question.
Many of the difficulties in cytokine gene identification mentioned above have been overcome by employing methods detailed in the present specification. These methods were used to isolate a number of human cDNA fragments which may encode immunomodulatory molecules.
In one aspect, the present invention includes a substantially-isolated polynucleotide having a sequence encoding a human polypeptide having immunomodulatory activity. In one embodiment, the polynucleotide has the sequence represented as SEQ ID NO:65. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:66. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:67. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:68. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:70. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:71. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:72. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:73. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:74. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:76. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:78. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:79. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:82. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:83. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:85. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:86. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:88. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:92. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:95. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:98. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:99. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:100. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:104. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:105. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:106. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:107. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:108. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:109. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:112. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:113. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:114. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:115. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:124. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:130. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:132. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:133. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:134. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:135. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:136. In another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:137. In yet another embodiment, the polynucleotide has the sequence represented as SEQ ID NO:138.
In a preferred embodiment, the polynucleotide contains a sequence selected from the group represented by SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:99, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:124, SEQ ID NO:130, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137 and SEQ ID NO:138. In another preferred embodiment, the polynucleotide contains a sequence selected from the group represented by SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78 and SEQ ID NO:79.
In another aspect, the present invention includes a substantially isolated human polypeptide having immunomodulatory activity, where the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:65. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:66. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:67. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:68. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:70. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:71. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:72. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:73. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:74. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:76. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:78. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:79. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:82. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:83. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:85. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:86. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:88. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:92. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:95. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:98. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:99. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:100. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:104. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:105. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:106. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:107. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:108. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:109. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:112. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:113. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:114. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:115. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:124. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:130. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:132. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:133. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:134. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:135. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:136. In another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:137. In yet another embodiment, the polypeptide has a sequence encoded by a polynucleotide having a sequence represented by SEQ ID NO:138.
In a preferred embodiment, the polypeptide has a sequence encoded by a polynucleotide selected from the group consisting of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:99, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:124, SEQ ID NO:130, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137 and SEQ ID NO:138. In another preferred embodiment, the polypeptide has a sequence encoded by a polynucleotide selected from the group consisting of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78 and SEQ ID NO:79.
In another aspect, the present invention includes a substantially-isolated polynucleotide having a sequence encoding a human homologue of yeast RAD50. In one embodiment, the polypeptide contains a polypeptide sequence encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO:54 and SEQ ID NO:55.
In a related aspect, the invention includes a substantially isolated human homolog of yeast RAD50 polypeptide. In one embodiment, the homolog polypeptide contains a polypeptide sequence encoded by a polynucleotide sequence selected from the group consisting of SEQ ID NO:54and SEQ ID NO:55.
Yet another aspect of the present invention includes a substantially-isolated polynucleotide having a sequence encoding a human homologue of Drosophila melanogaster Septin-2. In one embodiment, the polypeptide contains a polypeptide sequence encoded by the polynucleotide sequence represented by SEQ ID NO:97.
In a related aspect, the invention includes a substantially-isolated human Septin-2 homolog polypeptide. In one embodiment, the homolog polypeptide contains a polypeptide sequence encoded by the polynucleotide sequence represented by SEQ ID NO:97.
Still another aspect of the present invention includes a method of identifying the presence of activated T-cells in a sample containing a plurality of different cell types. The method includes performing a polymerase chain reaction amplification, where an aliquot of the sample (or homogenate/fraction thereof) serves as an amplification target and where the amplification is done using an oligonucleotide primer pair capable of selective amplification of a polynucleotide fragment having the sequence represented as SEQ ID NO:151. The amplification reaction generates an amplification product having a specific size, and the size of the amplification product is determined. The presence of amplification product of an expected size is indicative of the presence of activated T cells in the sample. In one embodiment, the oligonucleotide primer pair consists of primers having sequences represented as SEQ ID NO:149 and SEQ ID NO:150. In another embodiment, the sample is derived from adult tissue.
The invention also encompasses a method of identifying sequences encoding polypeptides having immunomodulatory activity. The method includes (i) selecting, by direct selection using sequences specific for region 5q23-31 of human chromosome 5, cDNA fragments isolated from tissues or cells expressing cytokines, (ii) grouping the fragments into xe2x80x9cbinsxe2x80x9d, where each bin represents cDNA fragments corresponding to a single gene or genetic locus, the grouping performed by sequencing the fragments and/or mapping the fragments to longer sequences derived from region 5q23-31 of human chromosome 5, and (iii) analyzing the tissue specificity of expression of transcripts corresponding to the fragments (transcripts from the gene or locus which the fragments represent). In one embodiment, the first step (step (i)) is performed using cDNAs obtained from cell lines and/or tissues expressing cytokines, such as activated T-cells. In another embodiment, the first step is performed using cDNAs obtained from a chromosome 5-specific activated T-cell cDNA library in lambda gt10; which was constructed using a kit from Life Technologies, Inc. and is deposited at Genelabs Technologies, Inc., Redwood City. In another general embodiment, the analyzing of tissue-specific expression is carried out using sequence-specific primers in a polymerase chain reaction amplification reaction containing target nucleic acids derived from tissues or cell lines of interest. Examples of tissues which may be used in determining the tissue specificity of expression include total embryo, fetal liver, fetal brain, fetal muscle, placenta, adult heart, adult muscle, adult liver, adult brain, adult pancreas, adult kidney, adult aorta, adult spleen, adult testis, adult bone marrow, resting T-cells and activated T-cells.
The present invention also includes a method of obtaining full-length sequences of genes or loci identified as having immunomodulatory activity. The method includes selecting a desired sequence identified in Table 1 and using the sequence to isolate overlapping clones. In one embodiment, such overlapping clones are isolated using rapid amplification of cDNA ends (RACE) PCR with cDNA obtained from tissues or cell lines of interest or from a cDNA or genomic DNA library. In another embodiment, the overlapping clones are isolated by direct hybridization screening of a cDNA or genomic DNA library made from, for example, T-cells, a lymphoma or a leukemia.
Also included in the invention is a method of identifying proteins having immunomodulatory activity. The method includes obtaining a full-length coding sequence of a gene represented by a sequence presented in Table 1 (e.g., as described above) and cloning the sequence into a recombinant expression vector. The resulting vector is then used to express recombinant polypeptides in selected host cells, such as E. coli. 
The invention also includes a method of identifying small molecules that affect alter and/or modulate the activity of immunomodulatory proteins such as described above. The method includes assaying the effects of a polypeptide having immunomodulatory activity in the presence and absence of a test small molecule compound, and identifying the test compound as effective if the test compound is effective to significantly alter the effects of the polypeptide. In one embodiment, the small molecule compound is one of a plurality of such compounds present in a combinatorial library, such as one of a plurality of small molecules in a small molecule combinatorial library, or one of a plurality of peptides in a peptide combinatorial library.
These and other objects and features of the invention will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying drawings.